The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection for human immunodeficiency virus (HIV). HIV infection has been shown to result in suppressed thymopoeisis precluding regeneration of T lymphocytes in the periphery in the SCID mouse model. To further examine the role of the thymus in HIV infection and the influence of nef on early events in infection we have used the SIV/macaque model of AIDS. For this study we compared the effect of pathogenic (SIVmac239) and nonpathogenic (SIVmac239/nef-delete) molecular clones of SIV on the thymus during the first 50 days post-infection (pi). The thymus was examined by in situ hybridization, immunohistochemistry, double label immunohistochemistry and multiparameter flow cytometry to determine the localization and immunophenotype of infected cells and changes in percentages of thymocyte progenitors. In addition, a morphometric analysis of cell proliferation and programmed cell death (PCD) was performed using immunohistochemistry for the Ki67 nuclear proliferation antigen and in situ-end labeling, respectively. SIV antigen and nucleic acid were confined primarily to the medulla irrespective of viral inoculum. However, many more positive cells were present in animals inoculated with SIVmac239. Analysis of cell proliferation and PCD revealed no significant alterations in animals infected with SIVmac239/nef-delete compared to uninfected controls. In contrast, animals inoculated with the pathogenic molecular clone SIVmac239 had a significant increase in PCD in the cortex and medulla coincident with peak viral loads at 14 days pi. This was followed by a marked increase in proliferating cells, primarily in the cortex at 21 days pi. This was also associated with marked changes in the percentages of immature thymocytes. These